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. 2013 Mar 8;288(10):7387-98.
doi: 10.1074/jbc.M112.400754. Epub 2013 Jan 11.

Dual-site interactions of p53 protein transactivation domain with anti-apoptotic Bcl-2 family proteins reveal a highly convergent mechanism of divergent p53 pathways

Ji-Hyang Ha  1 , Jae-Sun Shin, Mi-Kyung Yoon, Min-Sung Lee, Fahu He, Kwang-Hee Bae, Ho Sup Yoon, Chong-Kil Lee, Sung Goo Park, Yutaka Muto, Seung-Wook Chi
Affiliations

Dual-site interactions of p53 protein transactivation domain with anti-apoptotic Bcl-2 family proteins reveal a highly convergent mechanism of divergent p53 pathways

Ji-Hyang Ha et al. J Biol Chem. .

Abstract

Molecular interactions between the tumor suppressor p53 and the anti-apoptotic Bcl-2 family proteins play an important role in the transcription-independent apoptosis of p53. The p53 transactivation domain (p53TAD) contains two conserved ΦXXΦΦ motifs (Φ indicates a bulky hydrophobic residue and X is any other residue) referred to as p53TAD1 (residues 15-29) and p53TAD2 (residues 39-57). We previously showed that p53TAD1 can act as a binding motif for anti-apoptotic Bcl-2 family proteins. In this study, we have identified p53TAD2 as a binding motif for anti-apoptotic Bcl-2 family proteins by using NMR spectroscopy, and we calculated the structures of Bcl-X(L)/Bcl-2 in complex with the p53TAD2 peptide. NMR chemical shift perturbation data showed that p53TAD2 peptide binds to diverse members of the anti-apoptotic Bcl-2 family independently of p53TAD1, and the binding between p53TAD2 and p53TAD1 to Bcl-X(L) is competitive. Refined structural models of the Bcl-X(L)·p53TAD2 and Bcl-2·p53TAD2 complexes showed that the binding sites occupied by p53TAD2 in Bcl-X(L) and Bcl-2 overlap well with those occupied by pro-apoptotic BH3 peptides. Taken together with the mutagenesis, isothermal titration calorimetry, and paramagnetic relaxation enhancement data, our structural comparisons provided the structural basis of p53TAD2-mediated interaction with the anti-apoptotic proteins, revealing that Bcl-X(L)/Bcl-2, MDM2, and cAMP-response element-binding protein-binding protein/p300 share highly similar modes of binding to the dual p53TAD motifs, p53TAD1 and p53TAD2. In conclusion, our results suggest that the dual-site interaction of p53TAD is a highly conserved mechanism underlying target protein binding in the transcription-dependent and transcription-independent apoptotic pathways of p53.

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Figures

FIGURE 1.
FIGURE 1.
Interaction of p53TAD2 with Bcl-XL. A, domain organization of p53 and sequence alignment of p53TADs from different species. p53 consists of N- and C-terminal transactivation domains (N-TAD and C-TAD), proline-rich domain (PR), DNA-binding domain (DBD), oligomerization domain (OD), and C-terminal domain (CTD). Φ and X indicate a bulky hydrophobic residue and any other residue, respectively. Overlaid two-dimensional 1H-15N HSQC spectra for 15N-labeled wild-type p53TAD (B) and mutant p53TAD (L22A/W23A) (C) in the absence (blue) or presence (red) of Bcl-XL. D, cross-peak intensity ratio (Ibound/Ifree) for wild-type p53TAD (blue) and mutant p53TAD (L22A/W23A) (red). E, chemical shift perturbations on wild-type p53TAD (blue) and mutant p53TAD (L22A/W23A) (red) induced by Bcl-XL binding. Weighted ΔCS values were calculated using the equation ΔCS = ((Δ1H)2 + 0.2(Δ15N)2)0.5. Resonances that disappeared upon binding are shown as gray bars.
FIGURE 2.
FIGURE 2.
Binding of p53TAD2 to diverse anti-apoptotic Bcl-2 family proteins. A, overlaid two-dimensional 1H-15N HSQC spectra for 15N-labeled Bcl-XL, Bcl-2, and Bcl-w in the absence (blue) or presence (red) of p53TAD2 peptide. Binding site mapping of p53TAD2 on the structures (B) and molecular surfaces (C) of the anti-apoptotic Bcl-2 family proteins. The residues of the anti-apoptotic Bcl-2 family proteins showing chemical shift changes of ΔCS > 0.08 ppm and the disappeared residues are colored in red. The residues showing the chemical shift changes of 0.03 ppm < ΔCS < 0.08 ppm are colored in yellow.
FIGURE 3.
FIGURE 3.
Competitive binding between p53TAD2 and p53TAD1 to Bcl-XL. A, overlaid two-dimensional 1H-15N HSQC spectra for 0.1 mm 15N-labeled mutant p53TAD (L22A/W23A) in the free state (blue) or in the presence of 0.2 mm Bcl-XL (red) and in the presence of 0.2 mm Bcl-XL and 1 mm p53TAD1 peptide (green). B, overlaid two-dimensional 1H-15N HSQC spectra for 0.1 mm 15N-labeled mutant p53TAD (L22A/W23A) in the free state (blue) or in the presence of 0.2 mm Bcl-XL (red) and in the presence of 0.2 mm Bcl-XL and 1 mm Nutlin-3 (green).
FIGURE 4.
FIGURE 4.
Measurement of the binding affinity of Bcl-XL with p53TAD2 peptide. A, two-dimensional 1H-15N HSQC spectra of 15N-labeled Bcl-XL upon titration with p53TAD2 peptide (0 mm, blue; 0.12 mm, orange, 0.24 mm, cyan; 0.36 mm, yellow; and 0.48 mm, red). B, binding curves for the NMR titration of Bcl-XL with p53TAD2 peptide. C, ITC data of a titration of Bcl-XL with p53TAD2 peptide to measure the binding affinity.
FIGURE 5.
FIGURE 5.
p53TAD2 peptide bound to Bcl-XL adopts an α-helix. A, amide proton region in the two-dimensional 1H-1H transferred NOESY spectrum of p53TAD2 peptide in the presence of Bcl-XL. B, summary of the short and medium range NOEs for p53TAD2 peptide in the presence of Bcl-XL. The thickness of the bar represents the relative intensity of NOEs (strong or weak), and the gray bar indicates ambiguous NOE due to overlap. C, 13Cα shifts calculated by subtracting standard random coil values from the experimental 13Cα chemical shifts. Three different random coil values from Schwarzinger et al. (46) (top panel), Tamiola et al. (47) (middle panel), and Zhang et al. (48) (bottom panel) are used, and residues 39–57 are shown for clarity. The HNCA spectrum was acquired at 5 °C with 0.3 mm p53TAD in the presence of 0.3 mm Bcl-XL in 20 mm Tris-HCl (pH 7.5), 150 mm NaCl, and 1 mm DTT.
FIGURE 6.
FIGURE 6.
Refined structural models of p53TAD2 in complex with anti-apoptotic Bcl-2 family proteins. The best energy structural models of Bcl-XL·p53TAD2 (A) and Bcl-2·p53TAD2 complexes (B) are shown. Bcl-XL and Bcl-2 proteins are colored gray and yellow, respectively, and p53TAD2 peptide is colored blue. The BH1, BH2, and BH3 motifs of Bcl-XL are labeled. C and D, structural comparison of the Bcl-XL·p53TAD2 peptide complex with the Bcl-XL·Bim BH3 peptide complex (PDB code 3FDL). p53TAD2 and Bim BH3 peptides are colored blue and yellow, respectively. Chemical shift perturbations of Bcl-XL induced by p53TAD2 peptide are mapped onto the molecular surface of Bcl-XL (red) (C). Positive and negative electrostatic potentials are colored in blue and red, respectively (D).
FIGURE 7.
FIGURE 7.
PRE-NMR analysis of Bcl-XL by paramagnetically labeled p53TAD2 peptide. The two-dimensional 1H-15N HSQC spectra for 15N-labeled Bcl-XL in the presence of equimolar MTSL-labeled Cys-p53TAD2 were acquired in the oxidized (blue) and reduced (red) state. Two different regions of the overlaid two-dimensional 1H-15N HSQC spectra are shown in A and B.
FIGURE 8.
FIGURE 8.
Mutational analysis on the interaction between p53TAD2 and Bcl-XL. Chemical shift perturbations on the Bcl-XL residues by binding of wild-type and mutant p53TAD2 peptides (D49A, I50K, E51K, W53A, and F54A). Weighted ΔCS values were plotted against the residue number of Bcl-XL. Resonances that disappeared upon binding are shown as gray bars.
FIGURE 9.
FIGURE 9.
Mimicry in the dual site recognition of Bcl-XL, MDM2, and CBP by p53TAD. Structural models of the Bcl-XL·p53TAD2 (A) and Bcl-XL·p53TAD1 (C) complexes were compared with the previously reported structures of CBP NCBD·p53TAD2 (PDB code 2L14) (B) and MDM2·p53TAD1 (PDB code 1YCR) (D) complexes, respectively. The p53TAD1 and p53TAD2 peptides are drawn as green and blue ribbon models, respectively, and the ΦXXΦΦ motif residues involved in binding are labeled.
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